An affinity ligand has a function of specifically binding to a particular molecule, and an affinity separation matrix prepared by immobilizing the ligand to a water-insoluble carrier is utilized for efficient separation and purification of a useful substance from biological components or recombinant cells including microorganisms and mammalians. An industrially utilized antibody affinity ligand includes, for example, a peptide ligand or a protein ligand derived from a microorganism such as protein A, protein G and protein L or consisted of a functional variant (analog substance) obtained by recombinant technology thereof; a recombinant protein ligand such as a camel single strand antibody and an Fc receptor of an antibody; and a chemosynthetic ligand such as a thiazole derivative. The antibody affinity ligand is used in purification of an antibody drug and the like. Since the antibody preparation has lower toxicity and higher specificity than chemicals, there is much demand for an antibody drug as an ideal pharmaceutical.
A monoclonal antibody as active pharmaceutical ingredients of an antibody drug is mainly expressed in a culture fluid as a recombinant protein using a mammalian cultured cell or the like, and purified to a high purity by several steps of chromatography and filtration process before formulation. An antibody drug includes not only a molecule generally called an antibody such as immunoglobulin G and an analog thereof, but also an Fc fusion protein (Fc-containing molecule) in which an Fc region of a constant region of an immunoglobulin molecule is fused to another functional protein or peptide. Antibody drugs are also prepared by purifying and formulating from recombinant microorganisms, secreted substances in the culture supernatant, or expressed substances in bacterial cell or periplasmic space.
Impurities such as aggregates of antibodies (a dimeric and multiple form of a monomer) which are formed or remains in the steps of culture, purification and formulation is a major cause of side effects, and it is an important issue to reduce the impurities on production of an antibody preparation. Here, a monomer is defined as a unit of a molecule of an antibody having a tetramer structure composed of two molecules of heavy chains (H chains) consisting of an Fc region of a constant region, and a variable region, and two molecules of light chains (L chains) consisting of a variable region. A multimer of the unit molecules is regarded as an aggregate, and thought to be a major cause of side effects of an antibody preparation.
Attempts to control suppression of production of the aggregate and remove the aggregate have been made by a complicated management technique and use of an additive in the steps of culture, purification and formulation. Especially, not only suppression of production of the aggregate, but also removal of the aggregate is important in the purification step. Thus, development of a simple and efficient technique for removing the aggregate has been required in the purification step.
Patterning of purification techniques by combining particular unit operations (making of a platform) is developed in the purification step of the antibody preparation. In the early purification step (recovery step), an antibody affinity separation matrix in which protein A is immobilized as a ligand on a water-insoluble carrier (protein A carrier) is widely utilized. A technique of adsorbing an antibody to the protein A carrier under neutral conditions, and eluting the antibody under acidic conditions is generally used. However, in the elution process, the antibody subjected to the acidic conditions tends to be denatured and form an aggregate. In general, impurities such as an aggregate are removed by a combination of ion exchange chromatography, hydrophobic interaction chromatography and the like, in the subsequent step of protein A chromatography step (Non-patent Document 1, Non-patent Document 2, Non-patent Document 3, Patent Document 5).
However, after protein A chromatography step, high content of the aggregate is resulted in lowering of yield of the objective monomeric substance (monomer) in the subsequent step of removing impurities. Thus, not only suppression of formation of an aggregate, but also removal of an aggregate is studied in the protein A chromatography step.
The protein A chromatography step is generally carried out with acidic elution. However, since the lower the elution pH is, the more the risk of formation of an aggregate is, the protein A ligand is modified by means of protein engineering, so that the antibody which requires pH elution as low as about pH 3 can also be eluted near pH 3.5 to 4 (Patent Document 1).
In addition, a method for improving resolution of an aggregate from monomer fraction is examined in the protein A chromatography step. That is to say, optimization of pH and ionic strength at the time of elution, as well as fractionation of the first half of the elution peak and the second half of the elution peak, and the like are proposed. Concretely, there are methods utilizing slight decrease of dissociation constant by contacting an antibody molecule polymerized with a protein A ligand with probability higher than that of an antibody molecule which is not polymerized as a characteristic of the protein A carrier, and utilizing separation mechanisms based on delicate adjustment of hydrophobicity (Patent Document 2, Patent Document 3, Patent Document 4). However, since these methods are difficult to be strictly controlled and have low resolution, these methods are not used as a general separation technique in production of antibody drug.
As described above, although the antibody affinity separation matrix exhibits high specificity to an antibody and is capable of improving the purity, the ability of separating a monomeric substance (monomer) and an aggregate is low even if the usage is strictly set. Thus, the antibody affinity separation matrix had limitation for removing an aggregate.